Project Summary Some of the intracellular parasites of the phylum Apicomplexa, such as Plasmodium falciparum and Toxoplasma gondii, are among the most important infectious agents affecting humans. Due to rampant drug resistance, toxicity and lack of activity against certain stages there is a dire need for new anti-apicomplexan drugs. Calcium signaling in these parasites deserves special attention as a potential drug target as it drives many essential events such as motility, invasion and egress and it includes proteins not found in mammalian cells, such as the family of calcium dependent protein kinases (CDPKs). We recently discovered that a particular Toxoplasma CDPK, TgCDPK3, is required for efficient egress, division, calcium homeostasis and establishment of a chronic infection in vivo, presumably by maintaining the normal physiological state in which the parasite functions. To understand the mechanisms behind these various phenotypes we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant parasites through a near system-wide phosphoproteomic approach. This analysis revealed 156 peptides representing 106 proteins that are phosphorylated in a TgCDPK3 dependent manner, with many of them related to motility, ion-homeostasis, and metabolism. As a complementary approach, we performed a biotinylation-based screen for interacting proteins and identified 13 putative TgCDPK3-associated proteins, of which 7 were also identified as less abundant in the phosphoproteome of the mutant strain. We hypothesize that regulation of the phosphorylation state of a specific network of proteins by enzymes such as TgCDPK3 and calcium dependent phosphatases, is essential for the completion of Toxoplasma?s lytic cycle. Consistent with this idea chemical inhibition of either TgCDPK3 or the phosphatase calcineurin disrupts parasite exit from the host-cell. The main goal of this proposal is to characterize what we refer to as the ?phospho-program? of the lytic cycle. To do this we will: 1) Determine the role of ten TgCDPK3 putative substrates during the lytic cycle by generating and characterizing mutant parasite lines of each and defining their cellular localization. 2) Identify the sequence of phosphorylation events and the stoichiometry of phosphorylation in the identified network of 156 phosphosites. We will do this using a highly innovative targeted-proteomic approach that allows very rapid and high coverage phosphoproteome analysis. Using various mutant strains we will also determine the relative contribution of several proteins that are part of the network themselves on the signaling cascade. 3) Determine the function and substrates of the calcium-dependent phosphatase calcineurin during egress by genetically disrupting it and using two complimentary approaches to define the proteins it regulates. In conjunction, these studies will provide an in depth understanding of how phosphorylation regulates the propagation of the pathogenic parasite Toxoplasma, which would undoubtedly reveal vulnerabilities in the parasite?s biology that can exploited for the development of new therapies.